Genome-wide local ancestry and evidence for mitonuclear coadaptation in African hybrid cattle populations

Summary The phenotypic diversity of African cattle reflects adaptation to a wide range of agroecological conditions, human-mediated selection preferences, and complex patterns of admixture between the humpless Bos taurus (taurine) and humped Bos indicus (zebu) subspecies, which diverged 150–500 thousand years ago. Despite extensive admixture, all African cattle possess taurine mitochondrial haplotypes, even populations with significant zebu biparental and male uniparental nuclear ancestry. This has been interpreted as the result of human-mediated dispersal ultimately stemming from zebu bulls imported from South Asia during the last three millennia. Here, we assess whether ancestry at mitochondrially targeted nuclear genes in African admixed cattle is impacted by mitonuclear functional interactions. Using high-density SNP data, we find evidence for mitonuclear coevolution across hybrid African cattle populations with a significant increase of taurine ancestry at mitochondrially targeted nuclear genes. Our results, therefore, support the hypothesis of incompatibility between the taurine mitochondrial genome and the zebu nuclear genome.


INTRODUCTION
Hybridization between divergent lineages results in an influx of new genetic variants which can improve the adaptive potential of animal and plant populations (Hedrick, 2013;Moran et al., 2021). It has long been used by breeders to generate livestock populations with specific phenotypic characteristics (Wu and Zhao, 2021). For example, crossbreeding between Asian and European domestic pigs, which diverged 1 million year ago, was used by 19 th -century European breeders as a strategy to improve the fertility of local landraces (Bosse et al., 2014;White, 2011).
Human-mediated crossbreeding between the humpless Bos primigenius taurus (B. taurus -taurine) and the humped Bos primigenius indicus (B. indicus -zebu), which diverged 150-500 kya (Chen et al., 2018;Wang et al., 2018;Wu et al., 2018), has also played a major role in shaping the genetic composition of many African cattle populations. In fact, recent nuclear genome studies have shown that cattle ancestry in Africa represents a mosaic shaped by admixture between the original substrate of locally adapted taurine cattle, which likely first came to Africa with people during the Neolithic period, and the more recently introduced South Asian zebu (Kim et al., , 2020. This process of admixture, which lasted around 2,000 years (Kim et al., 2020), led to the establishment of indigenous African cattle populations that are deeply rooted in rural African communities, forming an integral part of food production and cultural and religious activities throughout the continent (Van Marle-Kö ster et al., 2021). The establishment of these African cattle populations with their unique phenotypic adaptations has been influenced by specific livestock breeding practices. In addition, their biology has been influenced by adaption to savanna biomes, cultural preferences, the logistics of long-distance terrestrial and maritime trade networks encompassing southern Asia, Arabia, and North and East Africa (Boivin et al., 2014;Boivin and Fuller, 2009;Gifford-Gonzalez and Hanotte, 2011;Marshall, 1989), and the massive cattle replacements following the rinderpest panzootics of the late 19th century (Spinage, 2003).
Functional mismatches between the mitochondrial and nuclear genomes transmitted from two divergent parental lineages have been observed in many vertebrate populations (Hill, 2019;Hill et al., 2019). For example, recent studies on hybridization in cattle, hares, sparrows, and hominids have provided compelling evidence for mitonuclear incompatibilities (Kwon et al., 2022;Seixas et al., 2018;Sharbrough et al., 2017;Trier et al., 2014). These likely stem from the fact that the 37 genes located in vertebrate mitochondrial genomes (Boore, 1999) also rely on over one thousand coadapted nuclear genes that encode proteins and protein subunits essential to the efficient functioning of the mitochondrion (Blier et al., 2001;Rand et al., 2004;Sloan et al., 2018;Woodson and Chory, 2008). The most well-studied example of mitonuclear cooperation is the oxidative phosphorylation (OXPHOS) system, which consists of five protein complexes, four of which are chimeric-assembled using subunits encoded by both the nuclear and mitochondrial genomes (Allen, 2015;Isaac et al., 2018;Rand et al., 2004). Mitonuclear incompatibilities between distinct inter-and intraspecific evolutionary lineages can give rise to deleterious biochemical effects associated with reduced efficacy of OXPHOS protein complexes (Ballard and Melvin, 2010;Blier et al., 2001;Ellison and Burton, 2006;Ellison et al., 2008), which lead to lower ATP production (Ellison and Burton, 2006;Ellison et al., 2008;McKenzie et al., 2003McKenzie et al., , 2004 and increased levels of oxidative damage (Barreto and Burton, 2013;Du et al., 2017;Latorre-Pellicer et al., 2016;Pichaud et al., 2019).
Fixation of the T1 haplogroup in African cattle has been investigated recently. An approximate Bayesian computation approach using genome-wide nuclear SNP data from 162 East African cattle indicated that a model of male-mediated dispersal combined with mitonuclear interactions could explain the current patterns of bovine genomic diversity in this region (Kwon et al., 2022). Here, we examine discordance of uniparental and biparental genomic variation in African cattle and test the hypothesis that functional incompatibilities have arisen between the mitochondrial and nuclear genomes in hybrid cattle populations across the continent (Figure 1). To do this, we analyzed high-density SNP data encompassing the nuclear and mtDNA genomes (Illumina BovineHD 777K BeadChip) from 605 animals representing 18 African, Asian, and European breeds/populations and 174 complete bovine mitochondrial genomes. These data were used to characterize genome-wide local ancestry and systematically evaluate mitonuclear interactions, coadaptation, and functional mismatch in multiple genetically independent admixed African cattle populations.

RESULTS AND DISCUSSION
Complex mitonuclear genomic structure in African admixed cattle We first established the ancestry of the animals in our sample set using the BovineHD 777K BeadChip data. Filtering and quality control of the BovineHD 777K BeadChip resulted in 562,635 SNPs and 605 individual animals, retained for subsequent analyses (Table 1). Figure 2A shows a principal-component analysis (PCA) plot generated using SNP genotype data for Asian B. indicus, East and West African admixed B. indicus/ taurus, African B. taurus, and European B. taurus cattle. PC1 (58.4%) and PC2 (17.9%) account for the bulk of the variance and represent the splits between B. indicus and B. taurus and the African and European taurine lineages, respectively. The results of the genetic structure analysis using the fastSTRUCTURE program and an inferred number of clusters of K = 3 are shown in Figure 2B, which illustrates taurine and zebu autosomal genomic ancestry across individual East and West African admixed animals and breeds ( Figure S1 and Table S1). These results recapitulate, at higher resolution, the continent-wide patterns of admixture that iScience Article were previously observed using smaller panels of microsatellite and SNP markers (Decker et al., 2014;Hanotte et al., 2002).
After filtering of the 346 mtDNA SNPs on the BovineHD 777K BeadChip and identification of ancestry-informative SNPs that distinguish the taurine and zebu mtDNA genomes, a network of eight haplotypes was generated using 39 mtDNA SNPs and a total of 491 cattle (47 African taurine, 82 European taurine, 156 East African admixed, 136 West African admixed, and 70 Asian zebu). Figure 3A shows this haplotype network and demonstrates that all 339 African taurine and admixed cattle surveyed here possess the taurine mitochondrial genome. In this respect, animals with predominantly zebu ancestry and morphology in Africa represent an example of ''massively discordant mitochondrial introgression'' (Bonnet et al., 2017), most likely as a result of male-mediated gene flow and genetic drift through preferential dissemination of B. indicus genetic material by a relatively small number of Asian zebu cattle, most of which were bulls (Bradley et al., 1994;Loftus et al., 1994a). This scenario is strongly supported by the widespread dissemination of the B. indicus Y chromosome in African admixed and morphologically taurine cattle populations (Hanotte et al., 2000;Perez-Pardal et al., 2018).

Evidence for positive selection at taurine and zebu mitochondrial OXPHOS protein genes
To assess whether the fixation of taurine mitochondrial ancestry in African cattle could be influenced by mitonuclear incompatibilities, we tested whether bovid mitochondrial sequences possess signals of species-specific adaptation. To do this, we obtained high-quality full mtDNA sequences from public DNA sequence databases for 126 African taurine and 21 Asian zebu mitochondrial genomes and 25 mitochondrial genomes for animals from six additional Bos species (B. gaurus -gaur; B. frontalis -mithun; B. grunniens -domestic yak; B. mutus -wild yak; B. javanicus -banteng; and B. primigenius -aurochs) ( Table S5). Fixed nucleotide substitutions were identified and cataloged from alignments of the 13  (Table S2).
We further tested for positive selection at the 13 OXPHOS protein genes using the branch-site test of positive selection (Yang and Nielsen, 2002;Zhang et al., 2005) based on the nonsynonymous/synonymous rate ratio (u = dN/dS) with positive selection indicated by u > 1 (Table S3). Individual genes showing statistically significant evidence for positive selection are indicated in Figure 3B, which shows that eight of the 13 OXPHOS protein genes have been subjected to positive selection in either the taurine (CYB, ND1, ND2, ND3, ND4L, and ND5) or the zebu (ATP6, ATP8, and COX1) mitochondrial genomes and that two (COX3  iScience Article and CYB) have undergone positive selection in both mtDNA lineages. These results provide evidence for positive selection that could lead to functional differences between zebu and taurine mitochondrial DNA sequences. However, to conclusively determine if these functional differences exist, biochemical and structural analyses of taurine and zebu mitochondrial proteins and their cognate nuclear partners will be required (Du et al., 2017;Sharbrough et al., 2017;Wang et al., 2017).

Nuclear-encoded mitochondrially targeted genes exhibit signatures of coadaptation across admixed African cattle populations
We then assessed whether admixed African cattle populations also preferentially retain taurine ancestry at nuclear genes encoding products targeted to the mitochondrion and those that directly interact with biomolecules produced from the mitochondrial genome. To do this, we reconstructed the local genomic ancestry of East and West African admixed populations, Asian zebu, and African taurine using MOSAIC (Salter-Townshend and Myers, 2019). Three functional subsets of genes were used in this analysis (Table S6): 1) high-confidence ''high-mito'' genes (HMG) encoding proteins that directly interact with mtDNA-encoded protein subunits in OXPHOS and ribosomal complexes or that have functions in mtDNA replication (136 genes); 2) lower confidence ''low-mito'' genes (LMG), which encode proteins that localize to the mitochondrion (661 genes) but are not classified as part of the high-mito subset; and 3) ''non-mito'' genes (NMG) representing the bulk of the mammalian proteome that does not localize to the mitochondrion (16,383 genes). For each admixed population, the taurine and zebu local ancestry estimates were averaged across mitochondrion-targeted genes (the HMG and LMG subsets) and compared to local ancestry estimates from the genomic background (NMG); this produced deviations in taurine local ancestry for each of the three functional gene subsets. We also generated coancestry curve plots using MOSAIC to determine the estimated number of generations since the start of admixture ( Figure S2).
From the bootstrap analysis ( Figure 4A), we found that three of the ten African admixed breeds individually exhibit significantly more taurine ancestry for the HMG subset: NGAN (p = 0.0160), KETE (p = 0.0410), and EASZ (p = 0.0430). Using the nonparametric Wilcoxon signed-rank test across the ten admixed African populations, we also demonstrated that the HMG subset exhibited significant differences in mean taurine ancestries compared to the LMG subset (p = 0.0039) and the NMG subset (p = 0.0020). We also compared mean taurine ancestries for the LMG versus the NMG subsets; however, this did not produce a significant statistical test result (p = 0.2754).

Functional consequences of mitonuclear incompatibilities in admixed African cattle breeds
Previous studies have examined subchromosomal admixture and local ancestry in hybrid taurine/zebu animals (Barbato et al., 2020;Chen et al., 2018;Koufariotis et al., 2018;Mbole-Kariuki et al., 2014;McTavish and Hillis, 2014), and we extend this work to mitonuclear incompatibilities and coadaptation in admixed cattle populations. Using a high-density SNP genotyping array, ten different breeds were examined with genome-wide zebu ancestries ranging between 37% (Borgou) and 74% (Karamojong) and estimated dates for the start of admixture in each population extending from the 14 th to the 20 th century ( Figure S1 and Table S1). A consistent pattern of mitonuclear disequilibria was observed for the functional HMG subset within three breeds of admixed African cattle (EASZ, KETE, and NGAN) ( Figure 4A): African taurine local ancestry was uniformly higher for nuclear genes encoding proteins that directly engage with mitochondrial-encoded gene products to form multi-subunit complexes or that directly interact with mitochondrial DNA or RNAs; this subset encompasses genes that encode OXPHOS subunits, ribosomal proteins, tRNA synthetases, and DNA and RNA polymerases. In support of the hypothesis that functional incompatibilities exist between the taurine and zebu mitochondrial genomes, we also find compelling evidence that the two mtDNA lineages have been subjected to positive selection at ten of the 13 OXPHOS protein genes ( Figure 3B and Table S2).
These results add support to the findings of Kwon et al. (2022), where they propose that the genomic composition of African admixed cattle has been influenced by selection pressure against the Bos indicus iScience Article mitochondrion. Similarly, although the source population divergence is substantially less in admixed humans, these results are comparable to those obtained by Zaidi and Makova (2019), which support the hypothesis that mitonuclear incompatibilities can act as a driver of selection in admixed human populations. They observed significant enrichment of sub-Saharan African ancestry for HMG subset genes in an African American population with sub-Saharan African and European nuclear ancestry and predominantly sub-Saharan African mtDNA haplotypes. They also observed significant enrichment of Native American ancestry at HMG subset genes in a Puerto Rican population with Native American and European nuclear ancestry and predominantly Native American mtDNA haplotypes.
The functional HMG and LMG subsets containing 136 and 661 genes, respectively (Table S6), were used in the present study for the purpose of evaluating mitonuclear incompatibilities in admixed African cattle populations. However, it is also instructive to examine these genes in the context of recently published high-resolution surveys of African cattle genomic diversity and signatures of selection (Table S4). Some of the genes detected using selection scans and analysis of population differentiated copy number variation in admixed African cattle have biochemical functions and physiological outcomes that may be impacted by mitonuclear incompatibilities (Jang et al., 2021). Proteins encoded by zebu alleles at these nuclear loci and the proteins encoded by the taurine iScience Article mitochondrial genome may interact suboptimally. For example, the aspartyl-tRNA synthetase 2, mitochondrial gene (DARS2), and an HMG subset gene on BTA16, are in the region encompassed by selective sweeps detected separately in the EASZ breed and a composite sample of East African zebu cattle Taye et al., 2018). Inspection of the Cattle Gene Atlas (Fang et al., 2020) demonstrates that DARS2 is most highly expressed in spermatozoa and therefore functionally linked to sperm motility, which may provide an explanation for mitonuclear coevolution in admixed cattle at this locus. In other words, mismatch between proteins encoded by zebu alleles of the DARS2 gene and the taurine mitochondrial OXPHOS complex proteins could reduce male fertility and lead to positive selection for taurine DARS2 alleles in admixed populations. In addition, the mitochondrial ribosomal protein S33 gene (MRPS33), another HMG subset gene, was detected within a positively selected region on BTA4 when African cattle were compared to commercial European and Asian breeds  and in analyses of selective sweeps focused on the evolution of thermotolerance in African cattle populations (Taye et al., 2017). Again, in this case, we can hypothesize that incompatibilities exist between zebu MRPS33 alleles and taurine mitochondrial genes. This could impact metabolism, homeostasis, and heat tolerance, giving rise to selection pressure acting to increase taurine ancestry at the MRPS33 locus.
Agriculture in Sub-Saharan Africa relies on a diverse array of indigenous cattle breeds, many of which show marked resilience to harsh environments, climatic extremes, and infectious disease-adaptations that have been shaped by their dual taurine-zebu ancestry. Cattle breeding programs in Africa are currently poised to leverage this composite ancestry through genomic selection as a leapfrog technology to bypass conventional breeding to enhance resilience (e.g., via the superior thermotolerance of zebu cattle), production, health, and welfare traits and ultimately improve the livelihoods of smallholder farmers (Ibeagha-Awemu et al., 2019;Marshall et al., 2019;Mrode et al., 2019). Future genetic improvement programs in African cattle will therefore need to consider mitonuclear incompatibilities that could reduce the fitness of hybrid taurine/zebu breeds. Understanding these incompatibilities in hybrid cattle may also provide useful information for targeted editing of both the bovine mitochondrial genome and mitochondrially targeted genes in the nuclear genome (Klucnika and Ma, 2020;Tang et al., 2021). Finally, our results demonstrate that admixed African cattle populations can serve as comparative model systems for understanding the phenotypic consequences of mitonuclear interactions and adaptive and maladaptive genomic introgression in other mammals, including humans.

Limitations of the study
Although we provide support for the hypothesis that mitonuclear coevolution exists between the nuclear and mitochondrial genomes of hybrid African cattle populations, this work is necessarily limited by the number of populations sampled and the density of the SNP data used. In addition, the genomewide approach we used here is not directly amenable to gene-by-gene analyses, which could use whole-genome sequence datasets from large numbers of hybrid animals to directly identify incompatibilities between individual nuclear-and mitochondrial-encoded proteins.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: